International Journal of Nanomedicine
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Open Access Full Text Article
Functional design of pH-responsive folate-
targeted polymer-coated gold nanoparticles for
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drug delivery and in vivo therapy in breast cancer
Sneha Mahalunkar 1
Amit Singh Yadav 2
Mahadeo Gorain 2
Vinay Pawar 3,4
For personal use only.
Ranveig Braathen 5,6
Siegfried Weiss 3
Bjarne Bogen 5,6
Suresh W Gosavi 7
Gopal C Kundu 2
1
School of Basic Medical Science,
Savitribai Phule Pune University, Pune
411007, Maharashtra, India; 2Laboratory
of Tumor Biology, Angiogenesis and
Nanomedicine Research, National
Centre for Cell Science (NCCS), Pune
411007, India; 3Institute of Immunology,
Hannover Medical School, Hannover,
Germany; 4Department of Molecular
Bacteriology, Helmholtz Centre for
Infection Research, Braunschweig,
Germany; 5K.G. Jebsen Centre for
Influenza Vaccines Research, Institute of
Clinical Medicine, University of Oslo,
Oslo, Norway; 6Oslo University Hospital,
Oslo 0027, Norway; 7Department of
Physics, Savitribai Phule Pune University,
Pune 411007, Maharashtra, India
Correspondence: Gopal C Kundu
Laboratory of Tumor Biology,
Angiogenesis and Nanomedicine
Research, National Centre for Cell
Science (NCCS), Pune 411007, India
Tel +91 202 570 8104
Fax +91 202 569 2259
Email kundu@nccs.res.in
Suresh W Gosavi
Department of Physics, Savitribai Phule
Pune University, Pune 411007,
Maharashtra, India
Tel +91 202 569 2678
Fax +91 202 569 1684
Email swg@physics.unipune.ac.in
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ORIGINAL RESEARCH
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Background: Curcumin has been widely used owing to its various medicinal properties
including antitumor effects. However, its clinical application is limited by its instability, poor
solubility and low bioavailability. Folic acid (FA)-functionalized nanoformulations may
enhance the sustained release of an anticancer drug (curcumin) by tumor-specific targeting
to improve therapeutic benefit. This study aims to design a nanoconjugate (NC) comprised of
folate–curcumin-loaded gold–polyvinylpyrrolidone nanoparticles (FA–CurAu-PVP NPs) for
targeted delivery in breast cancer model systems.
Methods: We developed curcumin-loaded FA-functionalized Au-PVP NCs by layer-by-
layer assembly. The folic acid–curcumin Au-PVP NCs (FA–CurAu-PVP NCs) were char-
acterized by ultraviolet–visible spectra, Fourier transform infrared spectroscopy, X-ray
powder diffraction and thermogravimetric analysis. In vitro anticancer and antimigratory
effects of NCs were examined by performing MTT and wound migration assays. The in vivo
antitumor efficacy of NCs was investigated using a preclinical breast cancer orthotopic
mouse model.
Results: Curcumin (40 µg/mL) was loaded along with conjugation of folate onto Au-PVP
NPs to form FA–CurAu-PVP NCs. The size and charge of the NCs were increased gradually
through layer-by-layer assembly and showed 80% release of curcumin at acidic pH. The NC
did not show aggregation when incubated with human serum and mimicked an intrinsic
peroxidase-like property in the presence of 3,3ʹ,5,5ʹ-tetramethylbenzidine substrate. The
MTT data using these NCs showed efficient anticancer activity at lower doses in estrogen/
progesterone receptor (ER/PR)-negative cells compared with ER/PR-positive cells.
Furthermore, the NCs did not show cytotoxicity at the investigated concentration in human
breast epithelial and mouse fibroblast cell lines. They showed inhibitory effects on cell
migration and high antitumor efficacy in in vivo analysis.
Conclusion: These results suggest that folate-based tumor targeting using CurAu-PVP NCs
is a promising approach for tumor-specific therapy of breast cancer without harming normal
cells.
Keywords: curcumin, gold nanoconjugate, folic acid, breast cancer cell line, cytotoxic
activity
Introduction
Chemotherapy, although a major treatment, is associated with various side effects
as well as poor compliance, and this has boosted significant efforts to find a better
anticancer drug modality that uses natural herbal compounds.1,2 As a result,
attempts made by the National Cancer Institute have led to the identification of
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 Mahalunkar et al
natural compounds that exhibit chemopreventive activity.3
Curcumin, a polyphenol compound isolated from Curcuma
longa Linn, can sensitize tumor cells by passing through
their lipid membrane.4 Extensive experimental research
has clearly shown that native curcumin can modulate
molecular targets, thereby inducing cell apoptosis in
human tumor-derived cell lines.5–10 Therefore, we have
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selected curcumin as a model compound to inform readers
about advanced drug-delivery therapies and assist them in
exploring other untouched platforms in chemotherapeutic
prevention.
The major hurdles in assigning natural compounds
such as curcumin as chemopreventives are their low bioa-
vailability, in vivo stability, non-specific biodistribution
and non-specific targeting properties. To overcome such
issues, various target specific drug-delivery systems have
been designed, consisting of nanoparticles loaded with
drugs and specific biomarkers, and this is the major area
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of interest in this particular study.11,12 Nanocarrier-based
delivery systems enable passive targeting due to the leaky
vasculature of tumors because of differences in their pore
size which enable nanocarriers to target the tumor and
accumulate at the tumor site, thereby blunting their effect
on normal tissues. Under many circumstances, cancer
patients with few other pathophysiological conditions
show a non-specific distribution of nanoconjugates to mul-
tiple sites which, to some extent, affects the targeting
specificity to the tumor zone.13,14 For this reason, nano-
carriers conjugated with drugs can be designed for active
targeting using targeting ligands such as folic acid (FA),
which can recognize specific receptors overexpressed on
certain cancer cell types.
FA is a well-known vitamin for cell proliferation as
well as targeting specific biomarkers. FA-functionalized
formulations may enhance sustained release of an antic-
ancer drug (curcumin) at the cellular targets to improve the
therapeutic benefits. Folate-conjugated nanomaterials have
elevated interest in the field of cancer biology, mainly
focusing on diagnosis and targeted drug-delivery
protocols.15–17 Salmaso et al formulated targeted delivery
by covalently conjugating polyethylene glycol (PEG) to
FA at one end of a polymeric carrier which has a cyclo-
dextrin–curcumin complex at the other end. Even though
such nanoconjugate complexes entered the clathrin-inde-
pendent pathway leading to cell endocytosis through tar-
geting overexpressed folate receptors, they showed limited
cellular uptake; hence, further investigation is required in
terms of drug release efficacy (DRE).18
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Chemically engineered nanocarriers made up of various
materials can be formulated and tuned to act as efficient
drug-delivery vehicles. To achieve better targeting and drug
release ability, gold nanoparticles (AuNPs) have predomi-
nantly been used as nanocarriers.19 A literature survey
showed effective cytotoxic activity of curcumin when it is
attached to one of the side chains of soluble polymers such
as PEG, polyvinylpyrrolidone (PVP) and chitosan, while
the other chain is attached to an AuNP, which also tackles
its problems of poor solubility and non-specificity.20 We
have explored FA conjugation and loading of curcumin
onto PVP-functionalized AuNPs to target breast cancer
cells, which thereby were proven to impart high cell-killing
ability. Therefore, a state-of-the-art layer-by-layer (LBL)
assembly method for designing nanoconjugates on a solid
surface of AuNPs by adsorbing anticancer drug with an
oppositely charged polymer such as PVP, involving steric
stabilization, is attracting much attention.21 The LBL
assembly can be further enhanced by loading activated
folate onto the above-formed conjugate using carbodiimide
coupling chemistry. The cross-linking agents that help in
forming an amide bond via coupling reactions are 1-ethyl-3-
[3-dimethylaminopropyl]carbodiimide (EDC) and dicyclo-
hexylcarbodiimide (DCC). Manju and Sreenivasan
demonstrated conjugation of hyaluronic acid with curcumin
using DCC/DMAP as a catalyst and coupling agent.21 The
carboxyl group of EDC/N-hydroxysuccinimide (NHS)-
functionalized folate can be conjugated with amine-functio-
nalized AuNPs on which the anticancer drug curcumin is
loaded, as shown in Scheme 1.22
With this background, we hypothesize that we can
remove the barriers and limitations associated with classical
drug-delivery protocols by encasing an anticancer drug such
as curcumin with properties of a multi-drug-resistant mod-
ulator and a chemosensitizer on Au-PVP NPs. A target-
specific strategy is warranted to enhance the therapeutic
efficacy of drug-loaded nanoparticles by conjugating them
with extensively investigated epithelial cancer markers such
as folate. Thus, our study aims to develop curcumin-loaded
FA-functionalized Au-PVP NCs by LBL assembly, which
can augment the therapeutic effects and improve the clinical
outcomes of curcumin in breast cancer therapy.
Materials and methods
Materials
The chemical and biological compounds used in this
research are as follows: folic acid (FA) (C19H19N7O6),
International Journal of Nanomedicine 2019:14
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Scheme 1 Schematic representation of folicacid–curcumin–gold–polyvinylpyrrolidone nanoparticles (FA–CurAu-PVP NCs) with layer-by-layer assembly.
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N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydro-
chloride (EDC.HCl) (C8H17N3.HCl), N-hydroxysuccini-
mide (NHS) (C4H5NO3) and 3,3ʹ,5,5ʹ-tetramethylbenzidine
(TMB) were obtained from Sigma-Aldrich (Bangalore,
India). Curcumin crystalline (C21H20O6) was purchased
from Loba Chemie (Mumbai, India); acetone, dimethyl
sulfoxide (DMSO) and H2O2 from Thomas Baker.
Chloroauric acid (HAuCl4) and trisodium citrate
(Na3C6H5O7) were purchased from SRL and were used as
received. Polyvinylpyrrolidone [(C6H9NO)n] was collected
from CDH. Trypsin/EDTA, penicillin, streptomycin, MTT
reagent, propidium iodide, RNase, FBS, DMEM and L-15
media were obtained from Sigma-Aldrich (Bangalore,
India). All chemicals were of reagent grade and were used
without further purification.
Maintenance of cell lines
Human breast adenocarcinoma (MDA-MB-231 and
MCF-7), mouse fibroblast (L929) and human breast
epithelial (MCF 10A) cell lines were purchased from
American Type Culture Collection (ATCC, Manassas,
VA, USA). Mouse mammary carcinoma cell line (4T1)
was purchased from Caliper Life Sciences (USA). Cell
lines were maintained in L-15 (MDA-MB-231) and
DMEM (MCF-7, T47D, 4T1, MCF 10A and L929) sup-
plemented with 10% FBS, 100 units penicillin and
100 μg/mL streptomycin. Cells were subcultured at
70–80% confluence and incubated at 37°C in an incuba-
tor supplied with 5% CO2.
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Synthesis of bare AuNPs and PVP-coated
AuNPs
Several methods of gold synthesis exist, which produce
diverse characteristics in the final product. The method
devised by Brust et al23 showed extensive use of organic
phase synthesis concerning a double- or a single-phase
process, based on gold salt reduction by sodium citrate
as reported by Turkevitch et al,24,25 which was refined by
Frens,26 who produced spherical size-tunable nanoparti-
cles. The purified AuNPs thus obtained were treated with
PVP polymer, which forms a thin covering around the
AuNP and can now be used to conjugate with the activated
ligands or with another nanoparticle or anticancer agents.
Activation of FA and its conjugation with
CurAu NPs to form FA–CurAu-PVP NCs
FA was converted into activated folate to achieve success-
ful conjugation with the amine terminated nanoparticles.
The activation of FA was achieved by the previously
reported method with few modifications, as follows.27–29
In brief, 2 mg FA was dissolved in 20 mL DMSO to obtain
a clear solution. To the above solution, EDC/NHS (0.4 M/
0.1 M) mixture was added, which resulted in activation of
COOH groups in FA (Fa/EDC/NHS 2:1:1) and the result-
ing solution was continuously stirred for about 1 h.
Similarly, curcumin (1 mg/mL) was dissolved in acetone
and was added in the amine terminated Au-PVP NP solu-
tion in a dropwise manner with stirring and heating con-
tinuously for 3 h, followed by washing the pellet several
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 Mahalunkar et al
times to remove the traces of unattached curcumin. The
activated folate mixture prepared earlier was then added to
5 mL of amine terminated CurAu-PVP NPs solution. The
conjugated FA–CurAu-PVP NPs were washed multiple
times with deionized water followed by suspending the
pellet in PBS. The NC thus formed was stored at 4°C until
further use.
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Physicochemical characterization of drug-
loaded NPs
The functionalized AuNPs were characterized by ultra-
violet (UV)–visible spectra (Jasco V-670 spectrophot-
ometer) recorded at 200–800 nm, and Fourier-transform
infrared (FTIR) spectra (Jasco FT/IR 6100) were also
recorded. The X-ray diffraction (XRD) patterns of curcu-
min and FA–CurAu-PVP NCs were obtained using a
Bruker D8 Advance X-ray diffractometer equipped
with Cu Kα radiation source from 10 to 80°C.
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Thermogravimetric analysis (TGA) was carried out
with a Mettler Toledo TGA/DSC 1 Stare System.
FA–CurAu-PVP NCs were also measured by the dynamic
light scattering (DLS) technique using a Zetasizer Nano
ZS90 instrument (Malvern) with an He-Ne laser beam at
a wavelength of 633.8 nm. Transmission electron micro-
scopy (TEM) was used to examine the morphology of the
nanosystems (Tecnai G22).
Determination of drug loading efficiency
The drug loading efficiency (DLE) of curcumin was deter-
mined. Curcumin solution was mixed with Au-PVP NPs in
a 1:1 ratio and sealed in porous dialysis tube to be dialyzed
against PBS (1×, pH 7.4) at room temperature for 24 h.
The released drug concentration was monitored by record-
ing UV–visible spectra. The drug loading efficiency was
calculated as follows:
DLE ¼ ðDrug loaded at time t = Total drugÞ  100
In vitro release of curcumin
Two volumes of 5.0 mL of FA–CurAu-PVP NCs were
enclosed in two dialysis bags with a molecular cut-off of
10 kDa and placed separately in two beakers, one contain-
ing 150 mL of PBS (1×, blood pH level 7.4) and another
with acetate buffer (1×, acidic pH 5.3), with continuous
stirring at 100 rpm. At predetermined time intervals,
100 µL of sample was collected and replaced with an
equivalent volume of release buffer solution. The released
concentration of drug as a function of time was analyzed
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by UV–visible spectroscopy. The amount of drug released
and cumulative release percentage were calculated using
the following formula:
DRE ¼ ðDrug released at time t = Total drugÞ  100
Colorimetric response of FA–CurAu-
PVP NCs
To investigate the peroxidase-mimetic activity of
FA–CurAu-PVP NCs, a 1:1 ratio of TMB and H2O2 was
mixed with 100 µL of 1 µg/mL NC and 0.01 M HNO3
solution. Next, the mixture of solution was kept at 50°C in
a water bath for 30 min and then cooled to room tempera-
ture. Subsequently, color change photographs were
acquired immediately and UV–visible readings were
recorded at 655 nm.
Protein adsorption studies
The UV–visible and FTIR spectra, particle size and zeta
potential of NPs can vary after serum protein adsorption or
interaction. To evaluate this phenomenon, human serum
(HS) was mixed with FA–CurAu-PVP NCs in a 1:1 ratio
with a pipette tip to obtain a homogeneous mixture. The
mixture was incubated for 22 h at 4°C followed by several
steps of washing with deionized distilled water before
measurement.
Cell viability assay
The effect of drug-loaded FA–CurAu-PVP NCs on cell
viability in breast cancer was evaluated by the MTT
assay. In brief, human breast cancer cells (MCF-7 and
MDA-MB-231) and mouse breast cancer cells (4T1)
were seeded (2×104) in a 96-well tissue culture plate.
Cells were treated with FA–CurAu-PVP NCs (10, 20, 50,
100 and 200 µg/mL) for 24 h. After 24 h incubation under
experimental conditions, the derivatives in the medium
were removed and MTT (0.5 mg/mL) was added and
incubated for 4 h. Thereafter, the formazan crystals were
dissolved in 200 µL of isopropanol and the absorbance
was recorded immediately at 570 nm using an automated
microplate reader (EPOCH2; BioTek Instruments,
Highland Park, VT, USA). Cell viability was analyzed
using SigmaPlot 10.0 software. In separate experiments,
Human mammary epithelial cells, MCF 10A, and mouse
fibroblast cells (L929) were grown in 96-well plates and
the MTT assay was performed.
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Wound healing assay
The effect of FA–CurAu-PVP on breast cancer cell migra-
tion was examined by a wound healing assay. In brief,
monolayers of MDA-MB-231 and 4T1 cells were grown
in 12-well plates and wounds of uniform size were made
with a sterile 10 µL pipette tip. Then, cells were treated
with different concentrations of NCs (5–20 µg/mL for
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MDA-MB-231 and 2.5–10 µg/mL for 4T1 cells) in low-
serum media with 4% FBS. Wound photographs were
captured at T=0 and T=18 h for MDA-MB-231 cells and
at T=0 and T=6 h for 4T1 cells using a phase-contrast
microscope (Nikon). The area migrated was quantified
(Image-Pro Plus software), analyzed and represented as a
bar graph (SigmaPlot 10.0 software).
Determination of cell cycle by flow
cytometry
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The effect of FA–CurAu-PVP on cell cycle progression in
MDA-MB-231 cells was analyzed by fluorescence-acti-
vated cell sorting. In brief, MDA-MB-231 cells were
seeded in a 60 mm dish at a density of 2×105 cells/dish
and treated with NCs (20 and 50 µg/mL) for 24 h. After
treatment, cells were fixed overnight in 95% ethanol and
washed twice in chilled PBS, then the pellet was resus-
pended in 50 μL of RNase A (0.5 mg/mL) and incubated
for 20 min at 37°C. After that, 450 μL of propidium iodide
(50 μg/mL) was added. The proportion of cells in different
phases of the cell cycle was monitored by a BD
FACSCalibur instrument (BD Biosciences).
In vivo antitumor efficacy
All animal experiments were conducted as approved by
the Institutional Animal Care and Use Committee
(IACUC) of the National Centre for Cell Science
(NCCS, Pune, India), in compliance with the ‘Committee
for the Purpose of Control and Supervision of Experiments
on Animals’ (CPCSEA) guidelines. 4T1 (1×105) cells in a
mixture with Matrigel (1:1) (BD Biosciences) were
injected into the mammary fat pads of 6-week-old female
Balb/c mice. Once tumors had formed, mice were ran-
domly divided into three groups. The first group was left
untreated as a control, while the second and third groups
were treated intratumorally with curcumin and FA–CurAu-
PVP NCs, respectively (10 mg/kg body weight) for 2
weeks. Tumor length and breadth were measured at reg-
ular intervals using Vernier calipers. Tumor volume was
calculated using the formula:
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Mahalunkar et al
h i
V ¼ π=6 ðlxbÞ3=2
After the required time period, mice were killed and
tumors were removed, photographed, weighed and pre-
sented graphically.
Statistical analysis
The data are presented as mean ± SEM using SigmaPlot
10.0 software. The level of significance was calculated
using the unpaired Student’s t-test. A p value less than
0.05 (p<0.05) was considered statistically significant.
Results
Physiochemical characterization of FA–
CurAu-PVP NCs
At the start, AuNPs were synthesized by the Turkevitch
method and analyzed by UV–visible spectroscopy. AuNPs
were coated with PVP, followed by loading of curcumin
on Au-PVP NPs and activation of FA into folate, thereby
achieving functionalization of nanoparticles with the EDC
coupling reaction, and then conjugating it with activated
folate to form FA–CurAu-PVP NCs. The NP thus formed
was analyzed and confirmed by UV–visible spectroscopy
as well as by FTIR, as shown in Figure 1.
The unmodified AuNPs displayed characteristic sur-
face plasmon absorption at 533 nm; when they were
coated with a polymer PVP there was a shift in the peak
position (532 nm), suggesting that the AuNPs had been
successfully coated with PVP (Figure 1A). The UV–visi-
ble spectra for pure FA showed one broad absorption band
around 285 nm, corresponding to the л-л* transition of the
C–C bond, and another broad shoulder around 364 nm,
corresponding to the n-л* transition of the C–O bond in
the enone moiety of FA. The spectrum of functionalized
FA also showed two broad peaks but with a bathochromic
shift from 285 to 312 nm and 364 to 376 nm. Thus, the
shift in the spectra of FA before and after functionalization
suggests the successful conversion of FA to activated
folate (Figure S1). Curcumin-conjugated AuNPs showed
a surface plasmon band around 223 nm and 532 nm,
suggesting loading of curcumin onto Au-PVP NPs
(Figure 1A). This new red peak indicates that curcumin
interacted with Au-PVP NPs. Two absorption maxima
peaks, at 285 nm and 364 nm, were observed in the
UV–visible spectrum of folate-conjugated nanomaterials
elsewhere, and the same data can be used to confirm
covalent attachment of the folate with AuNPs. Thus, we
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Figure 1 Step-by-step synthesis of folicacid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs). (A) Ultraviolet–visible spectra of AuNPs and
AuNPs coated with PVP. (B) Fourier transform infrared spectra of variation in forming different layers of NCs. (C) X-ray diffraction (XRD) spectra of FA–CurAu-PVP NC.
Inset: XRD spectra of curcumin. (D) DTG result of FA–CurAu-PVP NCs.
observed three major peaks in the FA–CurAu-PVP NC
spectrum. Two of these three peaks were at 285 nm and
364 nm, indicating folate covalent bonds, and the other
absorption peak was at 435 nm, confirming Au-PVP NP
formation, thus suggesting successful bonding of the FA
ligand to CurAu-PVP NPs.30–32 FA–CurAu-PVP NCs
showed peaks at 273 nm, 285 nm, 364 nm and 435 nm,
respectively. The results also indicate the increase in the
size of the sample after modification and conjugation.
Comparing the spectral data of FA and FA–CurAu-
PVP NCs shows that both of them had the same unique
peaks at 285 nm and 364 nm, which is assigned to the
π→π* transition of the pterin ring of the FA molecule,
indicating that FA was successfully conjugated with
CurAu-PVP NPs (Figure 1A).33 To further testify the
occurrence and qualitatively analyze conjugation of FA
on CurAu-PVP NPs, the nanoconjugates were character-
ized by FTIR spectroscopy. In the pure FA and functiona-
lized FA, the characteristic peak at 1651 cm−1 is accredited
to the stretching vibration of –C=O groups. The peaks
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plotted at 1500–1600 cm−1 were the stretching vibrations
of C=C determined for the aromatic ring backbone
structure.34 Comparing the infrared spectrum of FA with
that of FA–CurAu-PVP NCs, both of them have intensity
absorptions plotted at 1429 cm−1, 2911 cm−1 and 2999
cm−1. The peak around 1508 cm−1 is assigned to N–H
bending vibration of the free amine group of folate on
FA–CurAu-PVP NCs. When FA was conjugated with
CurAu-PVP NPs, a shift in the peak position was noticed
with respect to FA, CurAu-PVP NP and FA–CurAu-PVP
NCs between 3000 cm−1 and 3600−1, which was ascribed
to the hydroxyl (OH) stretching bands (Figure 1B). These
results suggest successful conjugation of FA on CurAu-
PVP NPs to form FA–CurAu-PVP NCs.34,35 Figure 1C
shows the XRD pattern of FA–CurAu-PVP NCs, which
reveals a typical amorphous pattern that may be due to
AuNPs and a few characteristic peaks of curcumin in the
2θ range of 10≤2θ≤30 degrees, which indicates the distinct
crystalline structure of a hydrophobic drug. Characteristic
peaks of curcumin appeared at 8.90, 12.25, 14.55 17.24,
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 Dovepress Mahalunkar et al

23.33, 24.60 and 25.52 degrees (Figure 1C, inset).

Furthermore, the diffraction 2θ peaks observed in the
range of 20–30 degrees for curcumin match those in ear-
lier reports.36–40
To study the change in sample weight as a function of
temperature and to analyze its thermal stability, a very
important technique, namely thermogravimetric analysis
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(TGA), was used. As shown in the thermogram in

Figure 1D, CurAu-PVP NPs showed only one step of
weight % decrease, while FA–CurAu-PVP NCs showed
two steps of weight % decrease. The temperature equiva-
lent to the deep peak at each weight change step is more
prominently observed in the first derivative of the TGA
curve (DTG) with respect to temperature to obtain a DTG
thermogram. The temperature which gives the maximum
rate of weight % for a sample (seen as peaks in the DTG
thermogram) is considered as the degradation temperature
of a component in the material. DTG thermogram of
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CurAu-PVP NPs showed one step degradation at 1250C

with 99% weight loss which might be the degradation
temperature (Td) of curcumin linked Au-PVP NPs
whereas after conjugating FA to form FA-CurAu-PVP
NCs, temperature at 1140C and 1450C showed 95% and
97% weight loss where the second Td might be ascribed
to the degradation temperature of folic acid conjugated
with CurAu-PVP NPs. The above data also indicate no
weight gain in the samples, suggesting that the conjugate
was intact without oxidation, and this may be due to the
Figure 2 Size distribution and transmission electron microscopy images of (A)
inert nitrogen gas atmosphere. We can thus conclude that curcumin–gold–polyvinylpyrrolidonenanoparticles (CurAu-PVP NPs), (B) folic acid–
the conjugates were successfully formed as FA–CurAu- curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs).

PVP NCs, which are slightly more stable than CurAu-PVP

NPs, and this may be due to layering of FA molecules.
To ascertain the formation of nanoconjugates, we per-
formed DLS and zeta potential measurements and TEM
before and after conjugation with the drug curcumin as
well as with a biomarker FA, as shown in Table S1. The
TEM images showed discrete spherical outlines and mono-
dispersed size distribution (~250 nm) (Figure 2A and B,
inset) which were lower values compared with DLS mea-
surements. The nanoconjugate of 250 nm will be ideal for
passive tumor targeting as most solid tumors show a vas-
cular cut-off between 380 and 780 nm, as mentioned
earlier.4–11 The DLS result reveals the average hydrody-
namic diameter of AuNPs to be 36.85 nm and that of
AuPVP to be 62.50 nm. The increase in hydrodynamic
size of CurAu-PVP NPs (72.56 nm) compared to
PVP-coated AuNPs can be credited to partial cross-linking
between the particles during the conjugation process
(Figure 2A),3 whereas the hydrodynamic diameter of
FA-grafted NCs (~358.7 nm) compared to CurAu-PVP
NPs with a fairly low polydispersity is consistent with the
NHS–folate grafting (Figure 2B).12 The significant increase
in the particle size of the NC formulation can be attributed
to the increase in shell volume that resulted from FA incor-
poration onto the CurAu-PVP NP core.12 The zeta potential
obtained for bare AuNPs was −7.47 mV, which shows a
noticeable change after its successful coating with PVP
(−9.91 mV) (Table S1). PVP is a neutral and hydrophilic
polymer which stabilizes Au-PVP through hydrophilic
interaction and therefore is efficiently loaded with hydro-
phobic oxygen-rich curcumin to form CurAu-PVP NPs.5
Attachment of partly negatively charged NHS–folate to
amine-functionalized CurAu-PVP NPs not only results in
increasing negative potential value of FA–CurAu-PVP NCs

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 Mahalunkar et al
but also ensures that the formulations will repel each
other to enable long-term storage without particle
aggregation.3–10 Previous reports show that particles with
a positive charge favor opsonization followed by reticuloen-
dothelialsystem clearance. Hence, a negative zeta potential
of approximately −5 to −20 mV is an appropriate system to
arbitrate drug delivery at sites other than the reticuloen-
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dothelial system.8,9 A step-by-step increase in the absolute
negative value of the zeta potential of AuNPs with FA–
CurAu-PVP NCs by increasing the polymer, drug and folate
content indicates that all three components were solubilized
not only on the AuNPs but also in the periphery of NCs.
The more negative charge of the NC could be also due to
the carboxylate group of FA located on the surface of the
nanoparticle.13 The elemental composition from the energy-
dispersive X-ray spectroscopy (EDS) spectra shows the
maximum percentage of AuNPs and the remainder as C
For personal use only.
Figure 3 Drug loading and release of curcumin. (A) Ultraviolet–visible spectra of drug loading onto the nanoconjugate. (B) Cumulative percentage of curcumin released
from folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) at pH 7.4 and pH 5.3. (C) Curcumin released from FA–CurAu-PVP NCs at pH
5.3. (D) Curcumin released from FA–CurAu-PVP NCs at pH 7.4.
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and Cl may be due to polymer, folate and curcumin cap-
ping. The EDS spectra of FA–CurAu-PVP NPs reveal the
elemental composition of curcumin and FA, with a higher
percentage of Ca, H, O and N along with C, Cl and Au
proving the successful conjugation of FA and curcumin
onto AuNPs (Figure S2).
In vitro drug loading and release studies
The DLE of curcumin was gradual with respect to time
and was calculated to be 40 µg/mL of FA–CurAu-PVP
NCs, and the DRE in the initial hours (2 h) was found to
be 40–45% at blood pH level (pH 7.4) and 80% at acidic
pH level (pH 5.3), which were better results than proposed
previously (Figure 3A and B).1 The release profiles at pH
5.3 and pH 7.4 of curcumin are shown in Figure 3C and D.
The loaded curcumin was released within a period of 24 h;
maximum release was seen within 8 h at pH 7.4 and 6 h at
International Journal of Nanomedicine 2019:14
 Dovepress
pH 5.3, followed by a gradual slow release. This clearly
implies that curcumin was loaded successfully onto
Au-PVP NPs. Thus, the goal of integrating an anticancer
drug (curcumin) onto the nanoparticle, along with FA, to
enhance its application in targeted drug-delivery protocols
was achieved.
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Serum binding studies
Analysis of FA–CurAu-PVP NCs in PBS before and after
incubation in HS by UV–visible spectra provided addi-
tional evidence about the protein coating. The presence of
such a protein coating layer on the nanoconjugates is also
known to prevent aggregation of the nanoparticles.11
Curcumin shows intense absorption from 200 to 450 nm
with maximum absorption close to 420 nm. Furthermore,
the UV–visible absorption spectra of curcumin bound to
HS have a shoulder peak around 450 nm, which is present
For personal use only.
in the spectra of FA–CurAu-PVP NC as well (Figure 4A).6
However, in the presence of HS, the curves of absorption
Figure 4 Serum binding studies. (A) Ultraviolet–visible spectra of folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) before and after
incubation with human serum and (B) Fourier transform infrared spectra of naive human serum and FA–CurAu-PVP NCs before and after incubation with human serum. (C)
Zeta potential measurements of FA–CurAu-PVP NCs before and after incubation with human serum. (D) Dynamic light scattering measurements of FA–CurAu-PVP NCs
before and after incubation with human serum.
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Mahalunkar et al
of curcumin show striking changes and this may be due to
the change in particle size because of protein binding.3,4
These changes indicate adsorption of serum proteins onto
the nanoconjugates. The addition of HS can promote the
movement of curcumin from a hydrophilic to a hydropho-
bic environment, as the aryl groups of curcumin are hydro-
phobically bound to the hydrophobic pockets of HS.5
Comparing the spectra of HS alone and when incubated
with FA–CurAu-PVP NCs gives information about differ-
ent types of secondary structures obtained after binding.4
The FTIR spectra of HS displayed a strong signal at
1645 cm−1, while the spectrum of FA–CurAu-PVP NCs
showed changes in the strength and position of the absorp-
tion peak between 1600 and 1700 cm−1 (Figure 4B). The
characteristic amide bands at about 1652, 1542 and 1241
cm−1 correspond to C=O stretching, N–H in-plane bend-
ing, and C–H stretching as well as C–N and N–H in-plane
stretching, respectively (Figure 4B).9,10 The changes in the
band intensity and slight shifts in infrared bands after
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 Mahalunkar et al
interaction with HS confirm the association of HS proteins
to FA–CurAu-PVP NCs. This suggests that by interacting
with FA–CurAu-PVP NCs, changes in the secondary
structure of HS protein have occurred.
To gain an insight into the HS-induced variation in
net surface charge and size of Au-PVP NPs, CurAu-
PVP NPs and FA–CurAu-PVP NCs, zeta potential and
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DLS measurements were carried out under standard
experimental conditions. Before incubation of these par-
ticles with HS, they were negatively charged. Upon
incubation, the mean particle surface charge increased;
that is, it became less negative, from −9.91 mV to −3.82
mV for Au-PVP NPs, from −10.1 mV to −5.09 mV for
CurAu-PVP NPs and from −12.5 mV to −3.74 mV for
FA–CurAu-PVP NPs (Figure 4C and Table S2). HS
protein binding to the FA–CurAu-PVP NCs is due to
electrostatic effects. Thus, the measured zeta potential of
the NC was highly negative before serum incubation
For personal use only.
and reached neutrality when bound with serum proteins.
It can be estimated that there is sequential binding of
cationic proteins initially, which may be covering the
negative charges on the particles, followed by opsoniza-
tion of negatively charged proteins to the positively
charged protein coat.3–8 In this study, we also observed
an increase in the size of AuNPs when incubated with
HS. The adsorbed serum proteins decreased the absolute
charge on the particles but increased the size and kept
them stable. This stability may be due to Coulombic
repulsion between charged particles or to steric hin-
drance caused by the protein coating, as stated
elsewhere.10 As illustrated in Figure 4D, the size of
nanoparticles almost doubles after their incubation with
HS, whereas the polydispersity index remains between
0.2 and 0.6, which demonstrates that the nanoparticles
are suitable for DLS (Table S2). Thus, DLS was able to
detect the increase in size due to enhanced serum pro-
tein attachment with the particles.1 According to pre-
vious investigations and the literature, degradation of
curcumin has shown that HS acts as an efficient trans-
porter for the curcumin molecule.7
Colorimetric assay
To analyze the peroxidase-mimetic activity of FA–CurAu-
PVP NCs, the catalytic oxidation of peroxidase substrate
TMB in the presence of a catalyst, H2O2, was tested. As
shown in Figure 5, the AuNPs catalyze TMB substrate oxi-
dation in the presence of H2O2 to produce a greenish blue
color complex. The maximum absorption peak appeared at
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655 nm (Figure 5A). The catalytic activity of NCs is also
dependent on the pH of the reaction buffer and incubation
temperature. Hence, the pH of the solution (1.0–3.0) and
temperature of the reaction system (40–60°C) were opti-
mized in this study. As shown in Figure 5C, the catalytic
activity reduced with increasing pH conditions, in the pH
range 1–3. Reduced activity was observed in neutral or basic
solutions compared to acidic solutions. The best result for a
color change from yellow to greenish blue was obtained at
pH 1.2 and temperature 50°C, with an HNO3 concentration
of approximately 220–230 µL. TMB has a structure similar
to diamine, which leads to a lower solubility in a weak basic
medium. Such pH-dependent catalytic activity is mainly
associated with the substrates and not with the NCs. On the
other hand, the activity elevates with increasing incubation
temperature. The experiments showed that the absorbance of
FA–CurAu-PVP NCs in the TMB–H2O2 system was much
higher at pH 1.2, and the same system at pH 1.0 and pH 2.2
showed slight absorbance at 655 nm (Figure 5C). The ele-
vated absorbance at 655 nm is considered to be the gradual
increase in the oxidation product of TMB.3 This result
demonstrated that both FA–CurAu-PVP NCs and H2O2
were required to catalyze TMB oxidation at standard pH
1.2. Thus, the result also confirmed that FA–CurAu-PVP
NCs exhibited an intrinsic peroxidase-mimetic activity,
mainly due to the presence of AuNPs in the nanoconjugate.
The inset in Figure 5C compares the formation of reaction
products against different pH within a time interval of 30 min
after the addition of the catalysts (FA–CurAu-PVP NCs and
HNO3) for the color change of TMB. The color change was
very fast as soon as the pH was adjusted to 1.2, and after the
addition of 0.01 M HNO3 the change from colorless to
greenish blue was observed. The maximum catalytic activity
occurred at pH 1.2 and temperature 50°C in the presence of
TMB–H2O2–FA–CurAu-PVP NCs in the ratio 1:1:1.
Therefore, FA–CurAu-PVP NCs show superior peroxidase-
like catalytic performance to the peroxidase substrate TMB
in the presence of H2O2 and under standard pH conditions.1
Each component separately treated with NC does not give
absorbance at 655 nm but the NC with all three components
(TMB, H2O2 and HNO3) shows the color change (Figure 5B
and D). The AuNPs can catalyze the TMB–H2O2 reaction,
where H2O2 is adsorbed on the surface of AuNPs and the O–
O bond of H2O2 may be broken up into the double HO˙
radical.4,5 Thus, the catalytic ability of AuNP is attributed to
the OH radical thus generated. The catalytic efficiency of the
NC is strongly dependent on the pH and temperature of the
reaction. The 0.01 M HNO3 was used in the reaction media to
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Figure 5 Colorimetric assay. (A) Ultraviolet (UV)–visible peaks showing the effect of different concentrations of HNO3. (B) Images illustrating color change after addition of
different concentrations of HNO3: (i) 20 μL, (ii) 30 μL, (iii) 50 μL, (iv) 100 μL, (v) 200 μL, (vi) 300 μL, (vii) 220 μL, (viii) 230 μL, (ix) 240 μL, (x) 260 μL, (xi) 280 μL, (xii)
300 μL. (C) UV-visible spectra depicting pH standardization to obtain color change. Inset: Color change at (i) pH 1.0 colorless, (ii) pH 1.2 greenish blue and (iii) pH 2.2
yellow. (D) Absorbance at 655 nm of different reaction components: (i) 3,3ʹ,5,5ʹ-tetramethylbenzidine nanoconjugates (TMB-NC), (ii) H2O2-NC, (iii) TMB-H2O2 and (iv)
folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs), TMB and H2O2.

achieve optimum pH conditions. Thus, the results showed
that the catalytic activity was faster in acidic pH solution and
this may be due to the presence of enough positive charges on
the NCs only in acidic conditions. As tumor tissues have a
more acidic environment than normal tissues, this peroxi-
dase-mimetic activity of the NC could be beneficial for
intelligent cancer treatments, which would not only kill
tumor cells by producing OH radicals but also relieve oxida-
tive stress in normal cells by reducing the H2O2 level.6
In vitro anticancer activity
To examine the in vitro anticancer activity of NCs, the
MTT assay was performed as described previously.41
Incubation of cancer cells (MDA-MB-231, MCF-7 and
4T1) with the NCs showed the efficient antitumor effect
of FA–CurAu-PVP NCs (IC50: 50 µg/mL) in both human
and mouse mammary cancer cells (Figure 6A–C).
Moreover, incubation of normal cell lines (L929 and
MCF 10A) with FA–CurAu-PVP NCs demonstrated low
cytotoxicity up to 50 µg/mL dose compared to cancer cells
(Figure 6D and E). However, higher doses (100 and
200 µg/mL) showed significant cytotoxicity in both can-
cerous and normal cells. Hence, the data suggest that NCs
can exhibit anticancer activity at low doses without harm-
ing normal cells. NCs exhibited a higher inhibitory effect
in estrogen receptor (ER)/progesterone receptor (PR)-
negative breast cancer cells (MDA-MB-231 and 4T1)
compared to ER/PR-positive breast cancer cells (MCF-7).
To further confirm our findings, we performed cell-cycle
analysis in MDA-MB-231 cells using flow cytometry. The

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Figure 6 In vitro antitumor effect of folicacid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) in breast cancer. (A–C) MCF-7, MDA-MB-
231 and 4T1 cells were seeded into 96-well plates and treated with increasing concentrations of FA–CurAu-PVP NCs (0–200 µg/mL) for 24 h. Cell death induced by
NCs was analyzed by MTT assay. The percentage inhibition of cell viability is represented graphically. Values are presented as mean ± SEM of three independent
experiments, (D, E) Mouse fibroblast cells (L929) and Human mammary epithelial (MCF 10A) were seeded into 96-well plates and treated with increasing
concentrations of FA–CurAu-PVP NCs (0–200 µg/mL) for 24 h. Cell death induced by NCs was analyzed by MTT assay. The percentage inhibition of cell viability
is presented graphically.

data showed an increase in the sub-G0 population in NC-
treated cells compared to controls, suggesting that NC
treatment induced cell death (Figure S3).
In vitro antimigratory potential of FA–
CurAu-PVP NCs
We further examined the antimigratory property of the
NCs in MDA-MB-231 and 4T1 cells. Cells grown in
monolayers were wounded and then treated with different
concentrations of NCs; they were then observed until a
specific time-point at which cell death did not occur. As
illustrated in Figure 7, the NCs significantly inhibited
migration of 4T1 and MDA-MB-231 cells even at very
low doses (Figure 7A and B), proving their antimetastatic
potential.
PVP NCs (10 mg/kg body weight) and the effect on
tumor growth was compared with untreated controls.
Tumor volumes were measured twice a week by
Vernier calipers (Figure 8A). At the end of the experi-
ment, mice were killed; tumors were removed, photo-
graphed and weighed (Figure 8A–D). The data
demonstrated that FA–CurAu-PVP NCs significantly
inhibited the 4T1 breast tumor growth compared to
controls. However, at the same dose curcumin did not
lead to any significant reduction in tumor growth com-
pared to controls. This may be due to low water solu-
bility, non-specific targeting and early clearance of
curcumin from the body. The above findings suggest
that FA–CurAu-PVP NCs enhanced the efficacy of cur-
cumin at a dose as low as 10 mg/kg body weight.

In vivo antitumor efficacy of FA–CurAu-
PVP in breast cancer
To examine the antitumor effect of FA–CurAu-PVP NCs
under in vivo conditions, 4T1 mouse mammary tumors
were generated in Balb/c mice. After tumor generation,
mice were treated with free curcumin and FA–CurAu-
Discussion
Nanotechnology has immense potential to produce major
advances in areas of enzyme immobilization, energy, food,
agriculture and medicine.42 Nanotechnology-based deliv-
ery systems can overcome shortcomings such as drug
solubility, permeability and early clearance issues asso-
ciated with small molecules and drugs, and can provide

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 Dovepress Mahalunkar et al
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Figure 7 Folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) inhibit migration of breast cancer cells. (A) Confluent monolayer of MDA-
MB-231 cells wounded with constant width and treated with FA–CurAu-PVP NCs (0–20 µg/mL) for 18 h. Photographs of wounds were taken at T=0 and 18 h. (B)
Confluent monolayer of 4T1 cells wounded with constant width and treated with FA–CurAu-PVP NCs (0–10 µg/mL) for 6 h. Photographs of wounds were taken at T=0 and
6 h. Migrated distances were measured using Image-Pro Plus software and analyzed statistically and presented graphically using SigmaPlot software. Values are presented as
mean ± SEM of three independent experiments.

smart therapeutic solutions such as tissue-specific che-
motherapy and phototherapy.43–46
In this study, an innovative targeted drug-delivery vehi-
cle (FA–CurAu-PVP NC) with a pH-responsive drug-
release system, favorable cytocompatibility and folate
receptor-mediated targeting is formulated via a simple cova-
lent cross-linking method for cancer therapy. In the present
study, folate-targeted nanoconjugates of CurAu-PVP NPs
were synthesized successfully for the delivery of curcumin.
Curcumin-loaded optimized FA–CurAu-PVP NCs were
spherical, with a mean particle size of approximately 250
nm estimated by DLS and TEM, a zeta potential of −12.5, a
narrow size distribution of 0.6 and curcumin DLE of
40 µg/mL. The NCs showed a sustained release behavior
during 48 h in two different pH conditions. In this study, we
also observed an increase in the size of AuNPs when
incubated with HS. The adsorbed serum proteins decreased
the absolute charge on the particles and kept them stable, as
proven by zeta potential and UV–visible spectra analysis.
According to previous investigations and the literature,
degradation of curcumin was observed to be reduced in
the presence of a serum protein in a buffer solution, which
also proves that HS acts as an efficient transporter for the
curcumin molecule. FA–CurAu-PVP NCs showed
enhanced peroxidase-like activity in the presence of perox-
idase substrate TMB with H2O2 as a catalyst under a
standard pH condition of 1.2.
The MTT assay and flow cytometry studies indicated the
higher potential of folate-modified NCs in the inhibition of
breast cancer cell proliferation and migration in vitro. Further,
the data demonstrated a higher cytotoxicity toward ER/PR-
negative compared to positive cells. This may be due to the
differential expression of FA receptors on ER/PR-negative and
positive cells. It has been reported that there is a higher
expression of folate receptors in triple-negative breast cancer,
justifying the higher anticancer activity of FA–CurAu-PVP in
triple-negative breast cancer cells.47,48 These findings thereby
establish the fact that the synthesized FA–CurAu-PVP NCs
showed prominent cytotoxicity in FA receptor-expressing
breast cancer cells at doses significantly lower than the doses

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Figure 8 Folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) suppress breast cancer growth under in vivo conditions. (A, B) 4T1 mouse
mammary cells were injected orthotopically into Balb/c mice and then 10 mg/kg body weight of curcumin and FA–CurAu-PVP NCs were injected intratumorally twice a
week for 2 weeks. Tumor volumes were measured twice a week using Vernier calipers, analyzed statistically and represented graphically (mean ± SEM, n=5; *p<0.006
compared to control tumor, **p<0.05 compared to curcumin-treated tumor). (C, D) Tumors were excised, photographed, weighed and analyzed statistically. The bar graph
represents mean tumor weights (mean ± SD, n=5; *p<0.05 compared to control).

at which they developed toxicity in normal cells (MCF 10A
and L929). The cytotoxic nature of these NCs can also be
explained by their enhanced cellular uptake via FA-mediated
endocytosis.5 The ability of cancer cells to undergo migration
and invasion has been designated as one of the hallmarks of
cancer.49 During the development of most human cancers,
primary cells move out and invade the neighboring tissues
and ultimately travel to distant sites to make new colonies,
leading to 90% of human cancer deaths. Hence, targeting
cancer cell migration and invasion is an important aspect of

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 Dovepress
cancer chemotherapy.41,50 Our data showed that FA–CurAu-
PVP NCs have excellent antimigratory potential in breast
cancer cells. Furthermore, FA–CurAu-PVP NCs significantly
inhibited breast tumor growth in an orthotopic mouse model.
The enhanced efficacy of curcumin in the present nanoformu-
lation may be the cumulative result of effective tumor target-
ing through FA receptors and prolonged slow release of the
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drug at the tumor site. Curcumin also showed higher solubility
in the present formulation.
These results suggest that the versatile folate-based
tumor targeting using CurAu-PVP NCs is a promising
candidate for chemotherapy to kill tumor cells without
harming normal cells.
Abbreviations
Au-PVP NPs, gold–polyvinylpyrrolidone nanoparticles; Cur,
curcumin; DLE, drug loading efficiency; DLS, dynamic light
scattering; ER, estrogen receptor; FA–CurAu-PVP NCs,
For personal use only.
folic acid–curcumin–gold–polyvinylpyrrolidone nanoconju-
gates; FA, folic acid; FTIR, Fourier-transform infrared; HS,
human serum; NC, nanoconjugate; PR, progesterone recep-
tor; TEM, transmission electron microscopy; TGA, thermo-
gravimetric analysis; TMB, 3,3ʹ,5,5ʹ-tetramethylbenzidine;
UV, ultraviolet; XRD, X-ray diffraction.
Acknowledgment
We would like to acknowledge the Department of Science
and Technology (DST), Government of India, for provid-
ing financial assistance under the project DST/IMRCD/
EU/Inno-Indigo/NanoCanTher to carry out this work.
Disclosure
The authors report no conflicts of interest in this work.
References
1. Naksuriya O, Okonogi S, Schiffelers RM, et al. Curcumin nanoformu-
lations: a review of pharmaceutical properties and preclinical studies
and clinical data related to cancer treatment. Biomaterials.
2014;35:3365–3383. doi:10.1016/j.biomaterials.2013.12.090
2. Ghosh S, More P, Derle A, et al. Diosgenin functionalized iron oxide
nanoparticles as novel nanomaterial against breast cancer. J Nanosci
Nanotechnol. 2015;15:9464–9472. doi:10.1166/jnn.2015.11704
3. Sporn MB, Suh N. Chemoprevention of cancer. Carcinogenesis.
2000;21:525–530. doi:10.1093/carcin/21.4.701
4. Fujisawa S, Atsumi T, Ishihara M, Kadoma Y. ROS-generation activity
and radical-scavenging activity of curcumin and related compounds.
Anticancer Res. 2004;24:563–570.
5. Simion V, Stan D, Gan AM, et al. Development of curcumin-loaded
poly (hydroxybutyrate-cohydroxyvalerate) nanoparticles as anti-
inflammatory carriers to human-activated endothelial cells. J
Nanopart Res. 2013;15:2108. doi:10.1007/s11051-013-2108-1
International Journal of Nanomedicine 2019:14
Powered by TCPDF (www.tcpdf.org)
Mahalunkar et al
6. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB.
Bioavailability of curcumin: problems and promises. Mol Pharm.
2007;4:807–818. doi:10.1021/mp700113r
7. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin:
preclinical and clinical studies. Anticancer Res. 2003;23:363–398.
8. Aggarwal BB. Apoptosis and nuclear factor-kappa B: a tale of asso-
ciation and dissociation. Biochem Pharmacol. 2000;60:1033–1039.
doi:10.1016/s0006-2952(00)00289-6
9. Thangapazham RL, Sharma A, Maheshwari RK. Multiple molecular
targets in cancer chemoprevention by curcumin. Aaps J. 2006;8:
E443–449. doi:10.1208/aapsj080352
10. Manju S, Sreenivasan K. Enhanced drug loading on magnetic nano-
particles by layer-by-layer assembly using drug conjugates: blood
compatibility evaluation and targeted drug delivery in cancer cells.
Langmuir. 2011;27:14489–14496. doi:10.1021/la202470k
11. Roy G, Shetti D, Yadav A, Kundu GC. Nanomedicine: therapeutic appli-
cations and limitations. In: Soni S, Salhotra A, Suar M, editors. Handbook
of Research on Diverse Applications of Nanotechnology in Biomedicine,
Chemistry, and Engineering. Hershey: IGI Global; 2015:1–28.
12. Panda JJ, Kaul A, Kumar S, et al. Modified dipeptide-based nano-
particles: vehicles for targeted tumor drug delivery. Nanomedicine
(Lond). 2013;8:1927–1942. doi:10.2217/nnm.12.201
13. Gerlowski LE, Jain RK. Microvascular permeability of normal and
neoplastic tissues. Microvasc Res. 1986;31:288–305. doi:10.1016/
0026-2862(86)90018-X
14. Siflinger-Birnboim A, Del Vecchio PJ, Cooper JA, Blumenstock FA,
Shepard JM, Malik AB. Molecular sieving characteristics of the
cultured endothelial monolayer. J Cell Physiol. 1987;132:111–117.
doi:10.1002/(ISSN)1097-4652
15. Patel K, Sundara BR, Chen Y, Lou X. Cytotoxicity of folic acid
conjugated hollow silica nanoparticles toward Caco2 and 3T3 cells,
with and without encapsulated DOX. Colloids Surf B Biointerfaces.
2016;140:213–222. doi:10.1016/j.colsurfb.2015.12.046
16. Das M, Sahoo SK, Fatouros D. Folate decorated dual drug loaded
nanoparticle: role of curcumin in enhancing therapeutic potential of
Nutlin-3a by reversing multidrug resistance. PLoS One. 2012;7:
e32920. doi:10.1371/journal.pone.0032920
17. Das M, Mohanty C, Sahoo SK. Ligand-based targeted therapy for
cancer tissue. Expert Opin Drug Deliv. 2009;6:285–304. doi:10.1517/
17425240902780166
18. Salmaso S, Barsani S, Semenzato A, Caliceti P. New cyclodextrin
bioconjugates for active tumour targeting. J Drug Target.
2007;15:379–390. doi:10.1080/10611860701349752
19. Manju S, Sreenivasan K. Gold nanoparticles generated and stabilized
by water soluble curcumin–polymer conjugate: blood compatibility
evaluation and targeted drug delivery onto cancer cells. J Colloid
Interface Sci. 2012;368:144–151. doi:10.1016/j.jcis.2011.11.024
20. Manju S, Sreenivasan K. Conjugation of curcumin onto hyaluronic
acid enhances its aqueous solubility and stability. J Colloid Interface
Sci. 2011;359:318–325. doi:10.1016/j.jcis.2011.03.071
21. Manju S, Sreenivasan K. Synthesis and characterization of a cyto-
toxic cationic polyvinylpyrrolidone-curcumin conjugate. J Pharm
Sci. 2011;100:504–511. doi:10.1002/jps.22278
22. Biju V. Chemical modifications and bioconjugate reactions of nano-
materials for sensing, imaging, drug delivery and therapy. Chem Soc
Rev. 2014;43:744–764. doi:10.1039/c3cs60273g
23. Brust M, Walker M, Bethell D, Schiffrin DJ, Whyman R. Synthesis
of thiol-derivatized gold nanoparticles in a two-phase liquid-liquid
system. J Chem Soc Chem Commun. 1994;7:801–802. doi:10.1039/
C39940000801
24. Werner S, Arthur F. Controlled growth of monodisperse silica spheres
in the micron size range. J Colloid Interface Sci. 1968;26:62–69.
doi:10.1016/0021-9797(68)90272-5
25. Turkevich J, Stevenson PC, Hillier J. The size and shape factor in
colloidal systems. J Discuss Faraday Soc. 1951;11:55. doi:10.1039/
df9511100055
submit your manuscript | www.dovepress.com
8299
DovePress
 Mahalunkar et al
26. Frens G. Controlled nucleation for the regulation of the particle size
in monodisperse gold suspensions. Nat Phys Sci. 1973;20:241.
27. Jia CJ, Schuth F. Colloidal metal nanoparticles as a component of
designed catalyst. Phys Chem Chem Phys. 2011;13:2457–2487.
doi:10.1039/c1cp21236b
28. Hyung BE, Young MY, Young-Hwan H. Characteristic optical
properties and synthesis of gold-silica core-shell colloids.
Scriptamaterialia. 2006;55:1127–1129.
29. Wang X, Yao S, Ahn HY, et al. Folate receptor targeting silica
International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 188.68.0.75 on 25-Oct-2019
nanoparticle probe for two-photon fluorescence bioimaging. Biomed
Opt Express. 2010;1:453. doi:10.1364/BOE.1.000453
30. Yang H, Zhuang Y, Xiaoxia HHD, et al. Silica-coated manganese
oxide nanoparticles as a platform for targeted magnetic resonance and
fluorescence imaging of cancer cells. Adv Funct Mater.
2010;20:1733–1741. doi:10.1002/adfm.200902445
31. Wu H, Liu G, Zhang S, et al. Biocompatibility, MR imaging and targeted
drug delivery of a rattle-type magnetic mesoporous silica nanosphere
system conjugated with PEG and cancer-cell-specific ligands. J Mater
Chem. 2011;21:3037–3045. doi:10.1039/c0jm02863k
32. Zhu Y, Fang Y, Kaskel S. Folate-conjugated Fe3O4–SiO2 hollow
mesoporous spheres for targeted anticancer drug delivery. J Phys
Chem. 2010;114:16382–16388.
33. Zhang Z, Jia J, Lai Y, Ma Y, Weng J, Sun L. Conjugating folic acid to
gold nanoparticles through glutathione for targeting and detecting
For personal use only.
cancer cells. Bioorg Med Chem. 2010;18:5528–5534. doi:10.1016/j.
bmc.2009.12.033
34. Mine E, Yamada A, Kobayashi Y, Konno M, Liz-Marzán LM. Direct
coating of gold nanoparticles with silica by a seeded polymerization
technique. J Colloid Interface Sci. 2003;264:385–390. doi:10.1016/
S0021-9797(03)00422-3
35. Amirthalingam T, Kalirajan J, Chockalingam A. Use of silica-gold core
shell structured nanoparticles for targeted drug delivery system. J
Nanomedic Nanotechnol. 2011;2:119. doi:10.4172/2157-7439.1000119
36. Gangwar RK, Dhumale VA, Kumari D, et al. Conjugation of curcumin
with PVP capped gold nanoparticles for improving bioavailability.
Mater Sci Eng. 2012;32:2659–2663. doi:10.1016/j.msec.2012.07.022
37. Patel A, Hu Y, Tiwari JK, Velikov KP. Synthesis and characterization
of zein-curcumin colloidal particles. Soft Matter. 2010;6:6192–6199.
doi:10.1039/c0sm00800a
38. Dey S, Sreenivasan K. Conjugating curcumin to water soluble poly-
mer stabilized gold nanoparticles via pH responsive succinate linker.
J Mater Chem. 2015;3:824–833. doi:10.1039/C4TB01731E
submit your manuscript | www.dovepress.com
8300
DovePress
Powered by TCPDF (www.tcpdf.org)
Dovepress
39. Anitha VG, Deepagan VV, Divya R, Menon D, Nair SV, Jayakumar
R. Preparation, characterization in vitro drug release and biological
studies of curcumin loaded dextran sulphate-chitosan nanoparticles.
Carbohydr Polym. 2011;84:1158–1164. doi:10.1016/j.carbpol.2011.
01.005
40. Gangware RK, Tomar GB, Dhumale VA, Sharma RB, Datar S.
Curcumin conjugated silica nanoparticles for improving bioavailabil-
ity and its anticancer applications. J Agric Food Chem.
2013;61:9632–9637. doi:10.1021/jf402894x
41. Kumar D, Haldar S, Gorain M, et al. Epoxyazadiradione suppresses
breast tumor growth through mitochondrial depolarization and cas-
pase-dependent apoptosis by targeting PI3K/Akt pathway. BMC
Cancer. 2018;18:52. doi:10.1186/s12885-018-4242-8
42. Dwevedi A, Routh SB, Yadav AS, Singh AK, Srivastava ON,
Kayastha AM. Response surface analysis of nano-ureases from cana-
valia ensiformis and cajanus cajan. Int J Biol Macromol.
2011;49:674–680. doi:10.1016/j.ijbiomac.2011.06.027
43. Guo B, Zhao J, Wu C, et al. One-pot synthesis of polypyrrole
nanoparticles with tunable photothermal conversion and drug loading
capacity. Colloids Surf B Biointerfaces. 2019;177:346–355. doi:10.
1016/j.colsurfb.2019.02.016
44. Wu C, Wang S, Zhao J, et al. Biodegradable Fe (III)–WS2–PVP
nanocapsules for redox reaction and TME-enhanced nanocatalytic,
photothermal, and chemotherapy. Adv Funct Mater. 2019;1901722.
doi:10.1002/adfm.v29.26
45. Yang H, Zhao J, Wu C, Ye C, Zou D, Wang S. Facile synthesis of
colloidal stable MoS2 nanoparticles for combined tumor therapy.
Chem Eng J. 2018;351:548–558. doi:10.1016/j.cej.2018.06.100
46. Prasad R, Chauhan DS, Yadav AS, et al. A biodegradable fluorescent
nanohybrid for photo-driven tumor diagnosis and tumor growth inhi-
bition. Nanoscale. 2018;10:19082–19091. doi:10.1039/c8nr05164j
47. Necela BM, Crozier JA, Andorfer CA, et al. Folate receptor-α
(FOLR1) expression and function in triple negative tumors. PLoS
One. 2015;10:e0122209. doi:10.1371/journal.pone.0122209
48. Zhang Z, Wang J, Tacha DE, et al. Folate receptor α associated with
triple-negative breast cancer and poor prognosis. Arch Pathol Lab
Med. 2013;138:890–895. doi:10.5858/arpa.2013-0309-OA
49. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.
Cell. 2011;144:646–674. doi:10.1016/j.cell.2011.02.013
50. Yadav AS, Pandey PR, Butti R, et al. The biology and therapeutic
implications of tumor dormancy and reactivation. Front Oncol.
2018;8:72. doi:10.3389/fonc.2018.00072
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Supplementary materials
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Figure S1 UV-Visible spectra representing activation of folic acid in o folate.

For personal use only.

A) B)

Figure S2 (A) EDS graph of AuNP, (B) EDS graph of FaCur @AuNC.

Figure S3 Effect FA@CurAu-PVP treatment on cell cycle in MDA-MB-231 cells was analyzed by flow cytometry. Briefly, 2×105 cells were seeded in 60 mm dish and treated
with 20 and 50 µg/ml of FA@CurAu-PVP for 24 hrs. Cells were stained with propidium iodide and analyzed by FACSCalibur (BD Bioscieces).

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Table S1 Hydrodynamic size (Dh), polydispersity index (PDI) and zeta potential of gold nanoparticles (AuNPs) and successively
functionalized AuNPs, curcumin–gold polyvinylpyrrolidone nanoparticles (CurAu-PVP NPs) and folic acid–curcumin gold–polyvinyl-
pyrrolidone nanoparticles (FA–CurAu-PVP NPs) at physiological pH
Sr. no. Formulation Dh (nm) PDI Zeta potential (mV)

1 AuNP 36.85 0.296 −7.47

2 AuPvP 62.50 0.338 −9.91
3 CurAu-PVP NP 72.56 0.582 −10
International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 188.68.0.75 on 25-Oct-2019

4 FA–CurAu-PVP NC 358.7 0.6 −12.5

Table S2 Summary of size (dynamic light scattering), polydispersity index (PDI) and charge of gold–polyvinylpyrrolidone (Au-PVP)
functionalized nanoparticles (NPs), curcumin–gold (CurAu NPs) and folic acid–curcumin gold (FA–CurAu) NPs before and after
incubation with human serum
Formulation DLS: hydrodynamic diameter (nm) PDI Zeta potential (mV)

Au-PVP NPs before incubation 62.5 0.338 −9.91

Au-PVP NPs after incubation 559 0.239 −3.82
CurAu NPs before incubation 72.5 0.582 −10.1
CurAu NPs after incubation 320 0.393 −5.09
For personal use only.

FA–CurAu NPs before incubation 358 0.6 −12.5

FA–CurAu NPs after incubation 657 0.393 −3.74

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